Agilent feature extraction software v9

Microarray analysis was performed by NIEHS molecular genomics core laboratory using the Agilent Whole Mouse Genome 4×44 multiplex format oligo arrays (014868) (Agilent Technologies) following the Agilent 1-color microarray-based gene expression analysis protocol. Starting with 500 ng of total RNA, Cy3 labeled cRNA was produced according to the manufacturer’s protocol. For each sample, 1.65 μg of Cy3 labeled cRNAs were fragmented and hybridized for 17 h in a rotating hybridization oven. Slides were washed and then scanned with an Agilent Scanner. Data were obtained using the Agilent Feature Extraction software (v9.5), using the 1-color defaults for all parameters. The Agilent Feature Extraction software performed error modeling, adjusting for additive and multiplicative noise. The resulting data were processed using the Rosetta Resolver system (version 7.2). In order to identify differentially expressed probes, ANOVA was used to determine if there was a statistical difference between the means of groups. The analysis was performed with setting parameters of at least 2-fold difference and significance at p < 0.01. Data were deposited in Gene Expression Omnibus (GSE138443).

Pulmonary function testing in healthy control and asthmatics (with medication withheld) was performed at the Severe Asthma Research Program (SARP) centers [13 (link)]. Studies were approved by each center’s Institutional Review Board including written informed consent.
Airway epithelial cells and alveolar lavage cells were obtained via bronchoscopy with epithelial brushings. Sample were prepared and microarrays were performed as described [12 (link),14 (link)]. Total RNA extracted from the samples was suspended in Qiazol solution using the QIACube system (QIAGEN Inc., Valencia, CA, USA). Complementary RNA (cRNA) labeled with the Cy5 fluorescent dye was hybridized to 4×44K v2 Whole Human Genome Microarrays and data were extracted using the Agilent Feature Extraction software v9.5 (Agilent Technologies Inc., Santa Clara, CA, USA).
Genomic DNA from whole blood was isolated using DNA purification kits (QIAGEN Inc., Valencia, CA, USA). Illumina HumanHap1M BeadChip or the Illumina HumanOmniExpress700k BeadChip was used for genotyping (Illumina, Inc., San Diego, CA, USA) [15 (link),16 (link)].